The female human reproductive cycle relies on a number of gonadotropin hormones. Principle among these are the pituitary hormones follicle stimulating hormone (FSH) and luteinizing hormone (LH). During oogenesis, the process by which the female germ cell, the ovum, is produced, occurs within a follicle. A follicle is a collection of cells in the ovary containing an oocyte (egg). Follicle maturation which ultimately leads to ovulation, is dependent on the stimulatory effects of FSH.
In each menstrual cycle, many follicles are recruited for the maturation of the oocytes. At the beginning of the approximately 28-day menstrual cycle the follicles are in the primordial form, which is simply an oocyte surrounded by a single layer of cells. As follicular growth and maturation is activated by FSH, multiple layers of granulosa cells form around the initial single layer of cells, a process that continues through to midcycle. These granulosa cells are responsible for nourishing the oocyte and for the production and release of estrogen. FSH, produced by the pituitary induces aromatase activity in the granulosa cells thereby increasing the production of estrogen. Thus, concurrent with the maturation of a follicle there is an increase in estrogen production in the early part of the 28-day menstrual cycle. The follicle also contains receptors for the second pituitary gonadotropin, LH. As the follicle continues to grow and mature by mid-cycle (approx. day 14), a space (antrum) develops inside the mass of granulosa cells. At mid-cycle a surge of LH production acts on LH receptors to cause the follicle to rupture and release the oocyte which travels down the fallopian tube and, which may subsequently be fertilized. The normal ovulating woman recruits approximately 300 immature oocytes for each menstrual cycle. During a normal cycle, all but one follicle will regress (atresia), and a single dominant follicle will emerge and go on to release an oocyte.
Ovulation induction (OI) and assisted reproductive technologies (ART) regimens are hormone regimens that comprise two main phases: a stimulatory phase and an ovulatory phase. When appropriate, a suppression phase may precede the stimulatory phase. The suppression phase involves the administration of a GnRH agonist, which is used to suppress LH or estradiol levels. The stimulatory phase starts with the administration of an agent having follicle stimulating activity (such as FSH), and is usually 6 to 10 days long. Typically, a patient is treated with about 150 IU of FSH per day starting at about three days after spontaneous or induced menstruation. FSH administration is continued until there is one follicle of mean diameter greater than or equal to about 16–18 mm (follicle development can be evaluated by ultrasound). At this point, the ovulatory phase is induced using a relatively large dose (5,000–10,000 IU) of an agent having LH activity, such as human chorionic gonadotrophin (hCG), to rupture the follicle and mimic the natural luteinizing hormone (LH) peak or surge that occurs mid-cycle, to trigger release of a single oocyte into the fallopian tubes. The patient is instructed to have intercourse 24 to 38 hours after administration of the large dose of hCG. During the stimulatory phase, administration of follicle-stimulating hormone (FSH), or an agent exerting follicle-stimulating activity, or an agent stimulating endogenous FSH release, stimulates ovarian follicular growth. Administration of FSH or an agent exerting FSH-like activity or an agent stimulating endogenous FSH release need not be continuous in the stimulatory phase, nor need it continue until the end of the stimulatory phase. Alternative agents that can act in either the stimulatory phase or the ovulatory phase are desirable, particularly orally available agents. US 2002/0103106 A1 (Palmer et al.) discloses the oral and subcutaneous use of inhibitors of type IV phosphodiesterase in the ovulatory phase, to trigger ovulation. US 2003/0018037 (Westbrook Lempriere et al.) discloses the use of PDE5 inhibitors after ovulation in a non-assisted cycle to improve embryo survival, increase birth weight, increase uterine blood flow and increase progesterone serum levels.
Ovulation induction (OI) is the treatment of anovulatory or ammenorheic women to cause the release of a single oocyte into the fallopian tubes for in vivo fertilization or intrauterine insemination (IUI). The goal of an OI regimen is to cause a single oocyte to be released, so as to avoid multiple pregnancies. In a conventional regimen for OI, the first phase of treatment is called the stimulatory phase.
In some patients undergoing OI it may be desirable to begin treatment with a suppression phase. In the suppression phase, pituitary gonadotrophins are suppressed by the administration of a gonadotrophin releasing hormone (GnRH) agonist prior to commencing therapy with FSH. Administration of a GnRH agonist is started in the luteal phase of a menstrual cycle (usually on about day 20 of a menstrual cycle). Suppression of ovarian function usually takes from 8 to 21 days with a GnRH agonist, and may be monitored by monitoring LH or estradiol (E2) levels (LH <5 IU/L, E2<50 pg/ml generally indicate adequate suppression). The stimulatory phase is then started by administration of FSH. The use of a GnRH agonist suppresses the natural LH peak or surge which can trigger the release of oocytes prematurely. This allows better timing of release of the oocyte, and consequently intercourse. In patients suffering from polycystic ovarian syndrome (PCOS), it is also desirable to use a GnRH agonist, because these patients often have inappropriately high endogenous LH levels, it also permits the suppression of LH throughout the stimulatory phase, permitting better response to FSH.
The stimulatory phase of OI is also often carried out using agents that provoke endogenous FSH release, such as clomiphene citrate or aromatase inhibitors. Clomiphene citrate is administered during a stimulatory phase (usually a dose of 50 to 100 mg on days 3 to 7 or 5 to 9 of the menstrual cycle), causing an increase in endogenous FSH secretion, leading to follicular growth. Aromatase inhibitors, for example Letrozole, Anastrozole, YM-511, may be given on days 3 to 7 or 5 to 9 of the menstrual cycle, and also provoke a release of endogenous FSH, as described in WO 02/083239, which is incorporated herein by reference.
In contrast to OI, where a single ovulatory follicle and a single oocyte is desired, in assisted reproductive technologies (ART) regimens, it is desired to collect as many oocytes in a single cycle as possible. Treatment of infertility by ART, such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), Gamete Intrafallopian Transfer Procedure (GIFT), and Zygote Intrafallopian Transfer Procedure (ZIFT), requires controlled ovarian hyperstimulation (COH) to increase the number of female gametes. Healy, et al., Lancet 343:1539–1544 (1994). For example, in vitro fertilization (IVF), which is now a commonly used treatment for human female and male subfertility, is a technique of ART based on retrieval of mature human oocytes followed by fertilization of the mature oocytes with spermatozoa. Under standard IVF treatment protocols, human mature oocytes are recruited by a long hormone treatment regimen, e.g. 30 days. This protocol is initiated by suppressing the patient's own FSH and LH by gonadotropin releasing hormone GnRH or an analog of GnRH, and is followed by injections of exogenous gonadotropins, e.g. FSH and/or LH, in order to ensure development of multiple preovulatory follicles. At an appropriate stage of follicular growth, multiple oocytes are harvested by aspiration immediately before ovulation. The aspirated oocyte is subsequently fertilized in vitro and cultured, typically for three days before transferral of the resulting embryo into the uterus at the 4–8 cell stage.
More specifically, standard regimens for COH in ART include a suppression phase, also called a down-regulation phase, in which endogenous LH is suppressed by administration of a GnRH agonist starting in the luteal phase of a menstrual cycle (usually on about day 20 of a menstrual cycle). Suppression of ovarian function usually takes from 8 to 21 days with a GnRH agonist, and may be monitored by monitoring LH or estradiol levels (LH<5 IU/L, E2<50 pg/ml generally indicate adequate suppression). Down regulation is followed by a stimulatory phase in which follicular development is induced by daily administration of follicle stimulating hormone (FSH), usually at about 75–600 IU/day.
Alternatively, a GnRH antagonist may be used, instead of a GnRH agonist, in which case follicular stimulation with FSH is started, usually on day 1, 2 or 3 after spontaneous or induced menstruation, and endogenous LH production is suppressed by administration of a GnRH-antagonist starting on about day 6 after menses. GnRH antagonist and FSH administration are continued until the criteria for administration of an ovulation-triggering dose of hCG are met, as described below.
During the stimulatory phase, the ovaries are examined by ultrasound, to detect and measure the developing follicles. Because multiple follicular development is the objective of COH protocols in ART, when the ovaries show at least 3 follicles with a mean diameter greater than 16 mm (preferably one of 18 mm), an injection of hCG (5,000–10,000 IU) is given to trigger ovulation. Oocyte recovery is timed for 36–38 hours after the hCG injection. Oocytes are usually recovered from pre-ovulatory follicles, by aspiration.
Despite the fact that protocols such as those described above have been used in clinical protocols for a number of years, these protocols are not without some significant disadvantages. FSH has a relatively short half life, and treatment with FSH for either OI or ART involves daily injections of relatively large doses of FSH (75–600 IU FSH daily) during the stimulatory phase. The daily injections can cause patient discomfort and inconvenience and can be relatively costly. Such large doses and daily administration has the related risk of producing ovarian hyperstimulation syndrome (OHSS), which in severe cases may be life-threatening. There are other additional side-effects from the gonadotropin preparations including weight gain, bloating, nausea, vomiting, the time involved with the monitoring process, and the unknown long-term cancer risk. These hormone treatment regimens will become even more of a problem when IVF is offered to perfectly normal women in these programs due to infertility problems associated with the males partner's poor sperm quality. Thus, there are problems associated with the current protocols used in oocyte generation for IVF. There remains a need for the production of greater quantities of oocytes that are amenable to ovulation.
Due to the risks involved with administration of gonadotropins, various alternative protocols have been suggested. One way to alleviate the risks, side effects, and economic disadvantages of controlled ovarian stimulation protocols involves the retrieval of immature oocytes followed by in vitro maturation. In this approach, the female is not stimulated, or receives only minimal stimulation, and the retrieved oocytes are subjected to hormonal treatment in vitro. This in vitro maturation (IVM) protocol involves a significant reduction/elimination in a number of the side effects mentioned above and has the secondary economic advantages of reducing the amounts of hormones used for the treatment. However, while in animals in vitro maturation (IVM) has become an efficient method for producing oocytes for IVF, the recorded success rates for clinical human IVM have been low.
As another alternative, replacement of FSH with alternative medicaments having the ability to aid follicular growth and which avoid the risks of OHSS would be highly desirable. Furthermore, the provision of a preparation which acts with FSH to cause follicular growth would also be highly desirable, as it could augment low endogenous FSH levels, causing follicular growth in those patients who are anovulatory due to low endogenous FSH levels, or it could augment exogenously administered FSH, permitting an improved response in poor responders in ART, or permitting the same response in ART with lower doses and/or less frequent injections of FSH, at the same time avoiding the risks of OHSS.